Immunogenicity

Administration of therapeutics can induce immune responses, which include the formation of anti-drug antibodies (ADA). Immunogenicity assessments are essential to the outcome and interpretation of safety, pharmacokinetic (PK) and pharmacodynamic (PD) data. The response to immunogenicity can range from no clinical effect to severe and life-threatening.

Immunogenicity assays are developed and validated for the accurate, reliable, and sensitive detection of anti-drug antibodies (ADA). Typically, immunogenicity assessment of a therapeutic product follows a tiered approach:

Screening:

Also referred to as the binding antibody (bAb) assay is used to perform the initial assessment of the clinical samples. This analysis is designed to detect all the potential ADA. Samples are scored as ADA reactive or ADA non-reactive.

Confirmation:

This phase of analysis evaluates specificity (to the therapeutic) of ADA that was detected in the screening phase. Samples are scored as ADA positive or ADA negative.

Characterisation:

Once samples have been screened for the presence of the ADA and the specificity of the ADA are confirmed, then the sample is subjected to the characterisation tier. This phase characterises the magnitude of the ADA response (titre) and whether the antibodies are neutralizing (nAb). nAb analysis is aimed at characterising the ability of the ADA to inhibit the activity (i.e. neutralise) of the therapeutic. nAb assays are developed and validated as a separate immunogenicity method. The parameters assessed are similar to that of the bAb method.

Synexa develops and validates bioanalytical methods for PK analysis according to the guidelines laid out by the following regulatory bodies:

European Medicines Agency (EMA):

Guideline on immunogenicity assessment of therapeutic proteins (2017).

Food and Drug Administration (FDA):

Immunogenicity testing of therapeutic protein products - developing and validating assays for anti-drug antibody detection (2019).

Method development:

The purpose of method development is to define and test the design of a method with a variety of conditions, before subjecting the method to validation. Parameters to be assessed during development include, but are not limited to the choice of assay platform, suitable reference standards, quality controls, matrix type, assay quantification range, and other assay conditions (such as required sample collection and storage conditions, incubation temperatures and duration, capture and detection molecule pairs and concentrations, wash cycles). Once these have been established the minimum required dilution (MRD) is determined.

Minimum required dilution is the sample dilution that results in a signal closest to assay diluent. This analysis determines the minimum dilution to which samples must be diluted to minimise the interference of any matrix components present in the samples.

Validation:

Bioanalytical method validation illustrates that an optimised method is suitable for the intended analysis of study samples. Synexa performs method validation according to the FDA and EMA guidelines.

Validated assays are subject to tests to assess the following criteria:

01

Accuracy and Precision (A&P):

Accuracy and Precision (A&P):

Accuracy and precision (A&P) defines the closeness of measured values to each other (precision) and the closeness to an expected nominal (accuracy). The A&P of a method needs to be illustrated across the detection range. A&P assessments aim to include positive control (PC) levels at low, medium, and high levels. A&P is determined by cumulative data from at least six independent experiments conducted over several days by more than one analyst.

02

Assay cut point (ACP):

Assay cut point (ACP):

This is defined as the signal response which designates samples as ADA reactive or ADA non-reactive. Statistical analysis is applied to the signal data of repeat analysis of naïve subject samples. One of three possible cut points, fixed, floating, or dynamic is selected based on statistical analyses.

03

Assay sensitivity and low positive control (LPC) determination:

Assay sensitivity and low positive control (LPC) determination:

All parameters in immunogenicity analysis are evaluated to reach maximum sensitivity. This is the lowest concentration of ADA that the assay can consistently, accurately, and reliably detect. Assay sensitivity is established, monitored, and assured by the inclusion of the calculated low positive control (LPC) with each analysis.

04

Specificity cut point (SCP):

Specificity cut point (SCP):

SCP determines the minimum level of drug-induced signal inhibition which must be displayed by a reactive sample to qualify as ADA positive. This parameter ensures that ADA samples are specific to the therapeutic. The SCP is applied during the confirmation tier of analysis.

05

Specificity:

Specificity:

Specificity assessment confirms the ability of the method to detect ADAs that bind to the biologic but not to other assay components (e.g., plates) or reagents. Specificity is assessed by demonstrating that binding is blocked by excess un-labelled biologic.

06

Spike and recovery:

Spike and recovery:

The accurate and reliable recovery of the analyte in matrix confirms that the assay is sufficiently selective for the ADA.

07

Selectivity:

Selectivity:

Selectivity assessments examine the ability of the assay to detect biologic-specific ADAs in a matrix, that may contain interfering substances. Selectivity can be assessed by measuring responses in drug-naive negative control samples.

08

Drug and/or Analyte tolerance:

Drug and/or Analyte tolerance:

The analysis of tolerance is strongly related to the sensitivity of the assay. Tolerance is a measure of the maximum level of free drug/analyte that can be present in samples, while still maintaining the required sensitivity to detect low level ADA responses. This information is critical to the analysis of the final immunogenicity results in conjunction with the PD and PK data.

Drug intolerance: Free drug present in the samples will bind to the ADA and prevent their detection by the assay. Additionally, free drug present in the samples will interfere with certain type of assay designs (binds to the coating or detection of the assay), and in so lead to false positive or false negative results.

Analyte or Target intolerance: Free target (functional ligand of the drug) present in the samples will bind to the drug used in the various steps of the assay (coating or detection solution) and in doing so prevent the detection of the ADA. This can lead to false positive or false negative results (depending on assay design).

09

Stability:

Stability: